Exfoliation syndrome (XFS), an age-related disorder which constitutes the most common identifiable cause of open-angle and angle-closure glaucoma, is characterized by the accumulation of an abnormal fibrillar material (XFM) in many intraocular tissues, particularly those structures that line the aqueous-bathed surfaces of the anterior segment. Deposits of XFM are composed of a complex mixture of extracellular matrix glycoproteins and proteoglycans assembled in a supramolecular fibrillar structure. Due to the highly diverse biochemical composition it is still not clear which are the primary elements involved in the formation of XF fibers and whether some of the deposited components or their immediate precursors are recruited to the lesions from the surrounding fluids (e.g. aqueous). Thus, despite all available information, there is still no data pointing out to specific candidate genes to support the generation of animal models. An understudied alternative approach is the development of a culture model that produces XF-like fibers in vitro, a potentially valuable paradigm that would allow the search for the pathogenetic pathway(s) leading to disease, conduct time-course kinetics of fibril assembly, supply fibrils for detailed biochemical analysis and be eventually amenable for pharmacologic interventions. In this sense, and as extensively illustrated in the Preliminary Studies section, we have obtained encouraging data from culturing Tenon's capsule fibroblasts from XFS patients in tri-dimensional systems, documenting the production of XF fibrils ultrastructurally similar, if not identical, to those observed in vivo. Based on our results we hypothesize that the use of selected XFS ocular cells in culture will provide a much needed model to study XF fibril assembly. To this end, we will take advantage of cutting-edge proteomic approaches, the availability of surgical specimens to establish a diversity of primary cultures from individuals with and without XFS, the accessibility to aqueous humors from the same cases, the newly developed Tenon's fibroblasts tri-dimensional cultures as well as previous technical experience acquired by the research group in culturing iris pigmented epithelial (IPE) cells to: (a) conduct proteomic analysis of XF-like fibrils produced in vitro by Tenon's fibroblasts from XFS cases;(b) verify whether cells obtained form IPE of XFS patients, cultured under various conditions, produce XF-like fibrils with the same EM characteristics and biochemical composition than Tenon's fibroblasts and/or XF fibrils produced in vivo;(c) identify common proteomic differences in the composition of the various culture supernatants of XFS vs. non-XFS controls and validate, through the parallel analysis of aqueous fluids from the same XFS and non- XFS controls, whether the observed differences might serve as prospective signature markers of the disease. The data generated by the proposed experiments will constitute the basis of a future R01 application. PUBLIC HEALTH RELEVANCE: Exfoliation (XF) syndrome is an age-related disorder which constitutes the most common identifiable cause of glaucoma worldwide. The extreme diversity of the molecules composing the XF lesions has so far precluded the creation of a much needed animal model of the disease. We propose to biochemically and ultrastructurally characterize a promising cell culture paradigm that produces XF-like fibers in vitro, which may prove to be a useful alternative approach to study the molecular basis of the disease and explore novel pharmacologic interventions.